N-of-1 medicine.

ABSTRACT: The fields of precision and personalised medicine have led to promising advances in tailoring treatment to individual patients. Examples include genome/molecular alteration-guided drug selection, single-patient gene therapy design and synergy-based drug combination development, and these approaches can yield substantially diverse recommendations. Therefore, it is important to define each domain and delineate their commonalities and differences in an effort to develop novel clinical trial designs, streamline workflow development, rethink regulatory considerations, create value in healthcare and economics assessments, and other factors. These and other segments are essential to recognise the diversity within these domains to accelerate their respective workflows towards practice-changing healthcare. To emphasise these points, this article elaborates on the concept of digital health and digital medicine-enabled N-of-1 medicine, which individualises combination regimen and dosing using a patient's own data. We will conclude with recommendations for consideration when developing novel workflows based on emerging digital-based platforms.

CURATE.AI COR-Tx platform as a digital therapy and digital diagnostic for cognitive function in patients with brain tumour postradiotherapy treatment: protocol for a prospective mixed-methods feasibility clinical trial.

INTRODUCTION: Conventional interventional modalities for preserving or improving cognitive function in patients with brain tumour undergoing radiotherapy usually involve pharmacological and/or cognitive rehabilitation therapy administered at fixed doses or intensities, often resulting in suboptimal or no response, due to the dynamically evolving patient state over the course of disease. The personalisation of interventions may result in more effective results for this population. We have developed the CURATE.AI COR-Tx platform, which combines a previously validated, artificial intelligence-derived personalised dosing technology with digital cognitive training. METHODS AND ANALYSIS: This is a prospective, single-centre, single-arm, mixed-methods feasibility clinical trial with the primary objective of testing the feasibility of the CURATE.AI COR-Tx platform intervention as both a digital intervention and digital diagnostic for cognitive function. Fifteen patient participants diagnosed with a brain tumour requiring radiotherapy will be recruited. Participants will undergo a remote, home-based 10-week personalised digital intervention using the CURATE.AI COR-Tx platform three times a week. Cognitive function will be assessed via a combined non-digital cognitive evaluation and a digital diagnostic session at five time points: preradiotherapy, preintervention and postintervention and 16-weeks and 32-weeks postintervention. Feasibility outcomes relating to acceptability, demand, implementation, practicality and limited efficacy testing as well as usability and user experience will be assessed at the end of the intervention through semistructured patient interviews and a study team focus group discussion at study completion. All outcomes will be analysed quantitatively and qualitatively. ETHICS AND DISSEMINATION: This study has been approved by the National Healthcare Group (NHG) DSRB (DSRB2020/00249). We will report our findings at scientific conferences and/or in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04848935.

Economic Evaluation Associated With Clinical-Grade Mobile App-Based Digital Therapeutic Interventions: Systematic Review.

BACKGROUND: Digital therapeutics (DTx), a class of software-based clinical interventions, are promising new technologies that can potentially prevent, manage, or treat a spectrum of medical disorders and diseases as well as deliver unprecedented portability for patients and scalability for health care providers. Their adoption and implementation were accelerated by the need for remote care during the COVID-19 pandemic, and awareness about their utility has rapidly grown among providers, payers, and regulators. Despite this, relatively little is known about the capacity of DTx to provide economic value in care. OBJECTIVE: This study aimed to systematically review and summarize the published evidence regarding the cost-effectiveness of clinical-grade mobile app-based DTx and explore the factors affecting such evaluations. METHODS: A systematic review of economic evaluations of clinical-grade mobile app-based DTx was conducted following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) 2020 guidelines. Major electronic databases, including PubMed, Cochrane Library, and Web of Science, were searched for eligible studies published from inception to October 28, 2022. Two independent reviewers evaluated the eligibility of all the retrieved articles for inclusion in the review. Methodological quality and risk of bias were assessed for each included study. RESULTS: A total of 18 studies were included in this review. Of the 18 studies, 7 (39%) were nonrandomized study-based economic evaluations, 6 (33%) were model-based evaluations, and 5 (28%) were randomized clinical trial-based evaluations. The DTx intervention subject to assessment was found to be cost-effective in 12 (67%) studies, cost saving in 5 (28%) studies, and cost-effective in 1 (6%) study in only 1 of the 3 countries where it was being deployed in the final study. Qualitative deficiencies in methodology and substantial potential for bias, including risks of performance bias and selection bias in participant recruitment, were identified in several included studies. CONCLUSIONS: This systematic review supports the thesis that DTx interventions offer potential economic benefits. However, DTx economic analyses conducted to date exhibit important methodological shortcomings that must be addressed in future evaluations to reduce the uncertainty surrounding the widespread adoption of DTx interventions. TRIAL REGISTRATION: PROSPERO International Prospective Register of Systematic Reviews CRD42022358616; https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022358616.

Characteristics of Mobile Health Platforms for Depression and Anxiety: Content Analysis Through a Systematic Review of the Literature and Systematic Search of Two App Stores.

BACKGROUND: Mobile health (mHealth) platforms show promise in the management of mental health conditions such as anxiety and depression. This has resulted in an abundance of mHealth platforms available for research or commercial use. OBJECTIVE: The objective of this review is to characterize the current state of mHealth platforms designed for anxiety or depression that are available for research, commercial use, or both. METHODS: A systematic review was conducted using a two-pronged approach: searching relevant literature with prespecified search terms to identify platforms in published research and simultaneously searching 2 major app stores-Google Play Store and Apple App Store-to identify commercially available platforms. Key characteristics of the mHealth platforms were synthesized, such as platform name, targeted condition, targeted group, purpose, technology type, intervention type, commercial availability, and regulatory information. RESULTS: The literature and app store searches yielded 169 and 179 mHealth platforms, respectively. Most platforms developed for research purposes were designed for depression (116/169, 68.6%), whereas the app store search reported a higher number of platforms developed for anxiety (Android: 58/179, 32.4%; iOS: 27/179, 15.1%). The most common purpose of platforms in both searches was treatment (literature search: 122/169, 72.2%; app store search: 129/179, 72.1%). With regard to the types of intervention, cognitive behavioral therapy and referral to care or counseling emerged as the most popular options offered by the platforms identified in the literature and app store searches, respectively. Most platforms from both searches did not have a specific target age group. In addition, most platforms found in app stores lacked clinical and real-world evidence, and a small number of platforms found in the published research were available commercially. CONCLUSIONS: A considerable number of mHealth platforms designed for anxiety or depression are available for research, commercial use, or both. The characteristics of these mHealth platforms greatly vary. Future efforts should focus on assessing the quality-utility, safety, and effectiveness-of the existing platforms and providing developers, from both commercial and research sectors, a reporting guideline for their platform description and a regulatory framework to facilitate the development, validation, and deployment of effective mHealth platforms.

Personalised, Rational, Efficacy-Driven Cancer Drug Dosing via an Artificial Intelligence SystEm (PRECISE): A Protocol for the PRECISE CURATE.AI Pilot Clinical Trial.

Introduction: Oncologists have traditionally administered the maximum tolerated doses of drugs in chemotherapy. However, these toxicity-guided doses may lead to suboptimal efficacy. CURATE.AI is an indication-agnostic, mechanism-independent and efficacy-driven personalised dosing platform that may offer a more optimal solution. While CURATE.AI has already been applied in a variety of clinical settings, there are no prior randomised controlled trials (RCTs) on CURATE.AI-guided chemotherapy dosing for solid tumours. Therefore, we aim to assess the technical and logistical feasibility of a future RCT for CURATE.AI-guided solid tumour chemotherapy dosing. We will also collect exploratory data on efficacy and toxicity, which will inform RCT power calculations. Methods and analysis: This is an open-label, single-arm, two-centre, prospective pilot clinical trial, recruiting adults with metastatic solid tumours and raised baseline tumour marker levels who are planned for palliative-intent, capecitabine-based chemotherapy. As CURATE.AI is a small data platform, it will guide drug dosing for each participant based only on their own tumour marker levels and drug doses as input data. The primary outcome is the proportion of participants in whom CURATE.AI is successfully applied to provide efficacy-driven personalised dosing, as judged based on predefined considerations. Secondary outcomes include the timeliness of dose recommendations, participant and physician adherence to CURATE.AI-recommended doses, and the proportion of clinically significant dose changes. We aim to initially enrol 10 participants from two hospitals in Singapore, perform an interim analysis, and consider either cohort expansion or an RCT. Recruitment began in August 2020. This pilot clinical trial will provide key data for a future RCT of CURATE.AI-guided personalised dosing for precision oncology. Ethics and dissemination: The National Healthcare Group (NHG) Domain Specific Review Board has granted ethical approval for this study (DSRB 2020/00334). We will distribute our findings at scientific conferences and publish them in peer-reviewed journals. Trial registration number: NCT04522284.

The proper connection between shelterin components is required for telomeric heterochromatin assembly.

Telomeric regions contain prominent sites of heterochromatin, which is associated with unique histone modification profiles such as the methylation of histone H3 at Lys9 (H3K9me). In fission yeast, the conserved telomeric shelterin complex recruits the histone H3K9 methyltransferase complex CLRC to establish subtelomeric heterochromatin. Although many shelterin mutations affect subtelomeric heterochromatin assembly, the mechanism remains elusive due to the diverse functions of shelterin. Through affinity purification, we found that shelterin directly associates with CLRC through the Ccq1 subunit. Surprisingly, mutations that disrupt interactions between shelterin subunits compromise subtelomeric heterochromatin without affecting CLRC interaction with shelterin component Pot1, located at chromosome ends. We further discovered that telomeric repeats are refractory to heterochromatin spreading and that artificial restoration of shelterin connections or increased heterochromatin spreading rescued heterochromatin defects in these shelterin mutants. Thus, subtelomeric heterochromatin assembly requires both the recruitment of CLRC by shelterin to chromosome ends and the proper connection of shelterin components, which allows CLRC to skip telomeric repeats to internal regions.

Tls1 regulates splicing of shelterin components to control telomeric heterochromatin assembly and telomere length.

Heterochromatin preferentially forms at repetitive DNA elements through RNAi-mediated targeting of histone-modifying enzymes. It was proposed that splicing factors interact with the RNAi machinery or regulate the splicing of repeat transcripts to directly participate in heterochromatin assembly. Here, by screening the fission yeast deletion library, we comprehensively identified factors required for telomeric heterochromatin assembly, including a novel gene tls1+. Purification of Tls1 and mass spectrometry analysis of its interacting proteins show that Tls1 associates with the spliceosome subunit Brr2. RNA sequencing analysis shows that the splicing of a subset of mRNAs are affected in tls1Δ cells, including mRNAs of shelterin components rap1+ and poz1+. Importantly, replacing rap1+ and poz1+ with their cDNAs significantly alleviated heterochromatin defects of tls1Δ cells, suggesting that the missplicing of shelterin components is the cause of such defects, and that splicing factors regulate telomeric heterochromatin through the proper splicing of heterochromatin factors. In addition to its role in telomeric heterochromatin assembly, Tls1-mediated splicing of shelterin mRNAs also regulates telomere length. Given that its human homologue C9ORF78 also associates with the spliceosome and is overexpressed in multiple cancer cell lines, our results suggest that C9ORF78 overexpression might alter the proper splicing of genes during cancer progression.

The proper splicing of RNAi factors is critical for pericentric heterochromatin assembly in fission yeast.

Heterochromatin preferentially assembles at repetitive DNA elements, playing roles in transcriptional silencing, recombination suppression, and chromosome segregation. The RNAi machinery is required for heterochromatin assembly in a diverse range of organisms. In fission yeast, RNA splicing factors are also required for pericentric heterochromatin assembly, and a prevailing model is that splicing factors provide a platform for siRNA generation independently of their splicing activity. Here, by screening the fission yeast deletion library, we discovered four novel splicing factors that are required for pericentric heterochromatin assembly. Sequencing total cellular RNAs from the strongest of these mutants, cwf14Δ, showed intron retention in mRNAs of several RNAi factors. Moreover, introducing cDNA versions of RNAi factors significantly restored pericentric heterochromatin in splicing mutants. We also found that mutations of splicing factors resulted in defective telomeric heterochromatin assembly and mis-splicing the mRNA of shelterin component Tpz1, and that replacement of tpz1+ with its cDNA partially rescued heterochromatin defects at telomeres in splicing mutants. Thus, proper splicing of RNAi and shelterin factors contributes to heterochromatin assembly at pericentric regions and telomeres.

Elimination of shelterin components bypasses RNAi for pericentric heterochromatin assembly.

The RNAi pathway is required for heterochromatin assembly at repetitive DNA elements in diverse organisms. In fission yeast, loss of RNAi causes pericentric heterochromatin defects, compromising gene silencing and chromosome segregation. Here we show that deletion of telomere shelterin components restores pericentric heterochromatin and its functions in RNAi mutants. We further isolated a separation-of-function mutant of Poz1 and revealed that defective telomere silencing, but not telomere length control, is critical for bypassing RNAi. Further analyses demonstrated that compromising shelterin-mediated heterochromatin assembly in RNAi mutants releases heterochromatin protein Swi6, which is redistributed to pericentric regions through RNAi-independent heterochromatin assembly pathways. Given the high mobility of Swi6 protein and that increased levels of Swi6 facilitates heterochromatin spreading as well as ectopic heterochromatin assembly, our results suggest that constitutive heterochromatin domains use multiple pathways to form high-affinity platforms to restrain Swi6, thus limiting its availability and avoiding promiscuous heterochromatin formation.

Epe1 recruits BET family bromodomain protein Bdf2 to establish heterochromatin boundaries.

Heterochromatin spreading leads to the silencing of genes within its path, and boundary elements have evolved to constrain such spreading. In fission yeast, heterochromatin at centromeres I and III is flanked by inverted repeats termed IRCs, which are required for proper boundary functions. However, the mechanisms by which IRCs prevent heterochromatin spreading are unknown. Here, we identified Bdf2, which is homologous to the mammalian bromodomain and extraterminal (BET) family double bromodomain proteins involved in diverse types of cancers, as a factor required for proper boundary function at IRCs. Bdf2 is enriched at IRCs through its interaction with the boundary protein Epe1. The bromodomains of Bdf2 recognize acetylated histone H4 tails and antagonize Sir2-mediated deacetylation of histone H4K16. Furthermore, abolishing H4K16 acetylation (H4K16ac) with an H4K16R mutation promotes heterochromatin spreading, and mimicking H4K16ac by an H4K16Q mutation blocks heterochromatin spreading at IRCs. Our results thus illustrate a mechanism of establishing chromosome boundaries at specific sites through the recruitment of a factor that protects euchromatic histone modifications. They also reveal a previously unappreciated function of H4K16ac in cooperation with H3K9 methylation to regulate heterochromatin spreading.

Hydration dynamics of a halophilic protein in folded and unfolded states.

Proteins from halophilic microorganisms thriving at high salinity have an excess of charged carboxylate groups, and it is widely believed that this gives rise to an exceptionally strong hydration that stabilizes these proteins against unfolding and aggregation. Here, we examine this hypothesis by characterizing the hydration dynamics of a halophilic model protein with frequency- and temperature-dependent (17)O magnetic relaxation. The halophilic protein Kx6E was constructed by replacing six lysine residues with glutamate residues in the IgG binding domain of protein L. We also studied the unfolded form of Kx6E in the absence of salt. We find that the hydration dynamics of Kx6E does not differ from protein L or from other previously studied mesophilic proteins. This finding challenges the hypothesis of exceptional hydration for halophilic proteins. The unfolded form of Kx6E is found to be expanded, with a weaker dynamical perturbation of the hydration water than for folded proteins.

Macromolecular crowding fails to fold a globular protein in cells.

Proteins perform their functions in cells where macromolecular solutes reach concentrations of >300 g/L and occupy >30% of the volume. The volume excluded by these macromolecules stabilizes globular proteins because the native state occupies less space than the denatured state. Theory predicts that crowding can increase the ratio of folded to unfolded protein by a factor of 100, amounting to 3 kcal/mol of stabilization at room temperature. We tested the idea that volume exclusion dominates the crowding effect in cells using a variant of protein L, a 7 kDa globular protein with seven lysine residues replaced by glutamic acids; 84% of the variant molecules populate the denatured state in dilute buffer at room temperature, compared with 0.1% for the wild-type protein. We then used in-cell NMR spectroscopy to show that the cytoplasm of Escherichia coli does not overcome even this modest (∼1 kcal/mol) free-energy deficit. The data are consistent with the idea that nonspecific interactions between cytoplasmic components can overcome the excluded-volume effect. Evidence for these interactions is provided by the observations that adding simple salts folds the variant in dilute solution but increasing the salt concentration inside E. coli does not fold the protein. Our data are consistent with the results of other studies of protein stability in cells and suggest that stabilizing excluded-volume effects, which must be present under crowded conditions, can be ameliorated by nonspecific interactions between cytoplasmic components.

Halophilic enzyme activation induced by salts.

Halophilic archea (halobacteriae) thrive in hypersaline environments, avoiding osmotic shock by increasing the ion concentration of their cytoplasm by up to 3-6 M. To remain folded and active, their constitutive proteins have evolved towards a biased amino acid composition. High salt concentration affects catalytic activity in an enzyme-dependent way and a unified molecular mechanism remains elusive. Here, we have investigated a DNA ligase from Haloferax volcanii (Hv LigN) to show that K(+) triggers catalytic activity by preferentially stabilising a specific conformation in the reaction coordinate. Sodium ions, in turn, do not populate such isoform and the enzyme remains inactive in the presence of this co-solute. Our results show that the halophilic amino acid signature enhances the enzyme's thermodynamic stability, with an indirect effect on its catalytic activity. This model has been successfully applied to reengineer Hv LigN into an enzyme that is catalytically active in the presence of NaCl.

Structural basis for the aminoacid composition of proteins from halophilic archea.

Proteins from halophilic organisms, which live in extreme saline conditions, have evolved to remain folded at very high ionic strengths. The surfaces of halophilic proteins show a biased amino acid composition with a high prevalence of aspartic and glutamic acids, a low frequency of lysine, and a high occurrence of amino acids with a low hydrophobic character. Using extensive mutational studies on the protein surfaces, we show that it is possible to decrease the salt dependence of a typical halophilic protein to the level of a mesophilic form and engineer a protein from a mesophilic organism into an obligate halophilic form. NMR studies demonstrate complete preservation of the three-dimensional structure of extreme mutants and confirm that salt dependency is conferred exclusively by surface residues. In spite of the statistically established fact that most halophilic proteins are strongly acidic, analysis of a very large number of mutants showed that the effect of salt on protein stability is largely independent of the total protein charge. Conversely, we quantitatively demonstrate that halophilicity is directly related to a decrease in the accessible surface area.

Protein stabilization and the Hofmeister effect: the role of hydrophobic solvation.

Using the IGg binding domain of protein L from Streptoccocal magnus (ProtL) as a case study, we investigated how the anions of the Hofmeister series affect protein stability. To that end, a suite of lysine-to-glutamine modifications were obtained and structurally and thermodynamically characterized. The changes in stability introduced with the mutation are related to the solvent-accessible area of the side chain, specifically to the solvation of the nonpolar moiety of the residue. The thermostability for the set of ProtL mutants was determined in the presence of varying concentrations (0-1 M) of six sodium salts from the Hofmeister series: sulfate, phosphate, fluoride, nitrate, perchlorate, and thiocyanate. For kosmotropic anions (sulfate, phosphate, and fluoride), the stability changes induced by the cosolute (encoded in m(3)=deltaDeltaG(0)/deltaC(3)) are proportional to the surface changes introduced with the mutation. In contrast, the m(3) values measured for chaotropic anions are much more independent of such surface modifications. Our results are consistent with a model in which the increase in the solution surface tension induced by the anion stabilizes the folded conformation of the protein. This contribution complements the nonspecific and weak interactions between the ions and the protein backbone that shift the equilibrium toward the unfolded state.

Anion modulation of the 1H/2H exchange rates in backbone amide protons monitored by NMR spectroscopy.

The ability of three anionic cosolutes (sulfate, thiocyanate, and chloride) in modulating the (1)H/(2)H exchange rates for backbone amide protons has been investigated using nuclear magnetic resonance (NMR) for two different proteins: the IGg-binding domain of protein L (ProtL) and the glucose-galactose-binding protein (GGBP). Our results show that moderate anion concentrations (0.2 M-1 M) regulate the exchange rate following the Hofmeister series: Addition of thiocyanate increases the exchange rates for both proteins, while sulfate and chloride (to a less extent) slow down the exchange reaction. In the presence of the salt, no alteration of the protein structure and minimal variations in the number of measurable peaks are observed. Experiments with model compounds revealed that the unfolded state is modulated in an equivalent way by these cosolutes. For ProtL, the estimated values for the local free energy change upon salt addition (m (3,DeltaG )) are consistent with the previously reported free energy contribution from the cosolute's preferential interaction/exclusion term indicating that nonspecific weak interactions between the anion and the amide groups constitute the dominant mechanism for the exchange-rate modulation. The same trend is also found for GGBP in the presence of thiocyanate, underlining the generality of the exchange-rate modulation mechanism, complementary to more investigated effects like the electrostatic interactions or specific anion binding to protein sites.

Influence of the Hofmeister anions on protein stability as studied by thermal denaturation and chemical shift perturbation.

The influence of external cosolutes on the thermal stability of the B1 domain of protein L (ProtL) has been studied by circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. The thermal denaturation midpoint is effectively modulated by the addition of a suite of anions and follows the Hofmeister series. The maximum increase in thermostability (corresponding to 14 degrees C) was observed in the presence of 1 M sodium sulfate. After conversion of the experimental data into the change in the virial coefficient, a mechanistic model was used to estimate the relative contributions from excluded volume and preferential anion solvation for each anion. As expected, the excluded volume term stabilizes the native conformation of ProtL for all the cosolutes, but opposite effects on protein stability arise from the anion's solvation depending on their tendency to interact with or to become excluded from the protein surface. This behavior is in agreement with the results of independent NMR experiments: the anions that strongly interact with the protein surface produce significant perturbations in the amide protein chemical shift (delta d23(HN)). A correlation obtained between delta d23(HN) and the temperature coefficients for the different amide protons provides qualitative information about the structural determinants for the interaction between the protein surface and the cosolute.